Cytochrome P450s and uses thereof

ABSTRACT

The invention relates to isolated cytochrome P450 polypeptides and nucleic acid molecules, as well as expression vectors and transgenic plants containing these molecules. In addition, the invention relates to uses of such molecules in methods of increasing the level of resistance against a disease caused by a plant pathogen in a transgenic plant, in methods for producing altered compounds, for example, hydroxylated compounds, and in methods of producing isoprenoid compounds.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/243,778, filed Apr. 2, 2014, which is a continuation of U.S.patent application Ser. No. 13/986,446, filed May 3, 2013 now issuedU.S. Pat. No. 8,722,363), which is a continuation of U.S. patentapplication Ser. No. 13/199,349, filed Aug. 26, 2011 (now issued U.S.Pat. No. 8,445,231), which is a continuation of U.S. patent applicationSer. No. 12/182,000, filed Jul. 29, 2008 (now issued U.S. Pat. No.8,263,362), which is a continuation of U.S. patent application Ser. No.10/097,559, filed Mar. 8, 2002 (now issued U.S. Pat. No. 7,405,057),which claims the benefit of U.S. Provisional Application Nos. 60/274,421and 60/275,597, filed on Mar. 9, 2001 and Mar. 13, 2001, respectively,all of which are hereby incorporated by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Apr. 1, 2014, is 65 kilobytes in size,and titled 207ESEQ001.txt.

FIELD OF THE INVENTION

This invention relates to cytochrome P450s and uses thereof.

BACKGROUND OF THE INVENTION

Cytochrome P450s encompass a superfamily of oxidases responsible for theoxidation of numerous endobiotics and thousands of xenobiotics. Inaddition, in plants, cytochrome P450s play important roles in woundhealing, pest resistance, signaling, and anti-microbial and anti-fungalactivity.

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by manySolanaceous species in response to a variety of environmental stimuli,including exposure to UV (Back et al., Plant Cell. Physiol. 389:899-904,1998) and infection by microorganisms (Molot et al., Physiol. PlantPathol. 379-389, 1981; Stolle et al., Phytopathology 78:1193-1197, 1988;Keller et al., Planta. 205:467-476, 1998). It is the primary antibioticor phytoalexin produced in tobacco in response to fungal elicitation,and it is derived from the isoprenoid pathway via its hydrocarbonprecursor, 5-epi-aristolochene (FIG. 1). Several of the biosyntheticenzymes leading up to 5-epi-aristolochene formation have been studied(Chappell, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:521-547, 1995),especially 5-epi-aristolochene synthase (BAS) (Vogeli and Chappell,Plant Physiol. 88:1291-1296, 1988; Back and Chappell, Proc. Natl. Acad.Sci. U.S.A. 93:6841-6845, 1996; Mathis et al., Biochemistry36:8340-8348, 1997; Starks et al., Science 277: 1815-1820, 1997). BAScommits carbon to sesquiterpene metabolism by catalyzing the cyclizationof farnesyl diphosphate (FPP) to 5-epi-aristolochene. However, until thepresent invention, the enzyme(s) responsible for the conversion of5-epi-aristolochene to capsidiol has yet to be fully identified andcharacterized.

Biochemical evidence from previous studies in tobacco (Whitehead et al.,Phytochemistry 28:775-779, 1989) and green pepper (Hoshino et al.,Phytochemistry 38:609-613, 1995) have suggested that the oxidation of5-epi-aristolochene to capsidiol occurs in a two step process with oneof the hydroxylation steps being constitutive and the other beingmediated by an elicitor-inducible cytochrome P450 (FIG. 1). Because1-deoxycapsidiol had been isolated from natural sources (Watson et al.,Biochem. Soc. Trans. 11:589, 1983), Whitehead et al. (Phytochemistry28:775-779, 1989), surmised that perhaps the biosynthesis of thisintermediate was due to pathogen induction of a correspondinghydroxylase. They therefore prepared synthetic 1-deoxycapsidiol andreported a modest conversion of this compound to capsidiol when fed tocontrol or unelicited tobacco cell cultures. This was further supportedby their observation that radiolabeled 5-epi-aristolochene was onlyconverted to capsidiol when fed to elicitor-induced cell cultures butnot control cultures. Whitehead et al. (Phytochemistry 28:775-779, 1989)therefore concluded that the 3-hydroxylase, responsible forhydroxylation of 5-epi-aristolochene at C3 to generate 1-deoxycapsidiol,was pathogen/elicitor inducible, while the 1-hydroxylase, responsiblefor hydroxylating 1-deoxycapsidiol at the C1 to generate capsidiol, wasconstitutive. Hoshino et al. (Phytochemistry 38:609-613, 1995) added tothe observations of Whitehead et al. (Phytochemistry 28:775-779, 1989)by directly measuring 3-hydroxylase-activity in microsomal preparationsof arachidonic acid-elicited Capsicum annuum fruits and seedlings. Theseassays consisted of incubating 5-epi-aristolochene with microsomepreparations and subsequently determining the amount of 1-deoxycapsidiolgenerated by a combination of thin-layer chromatography (TLC)separations and gas chromatography (GC). Their evidence demonstratedthat the conversion of 5-epi-aristolochene to 1-deoxycapsidiol wasdependent on both NADPH and O₂, and that 1-deoxycapsidiol accumulationin vitro was arrested by the P450 antagonists carbon monoxide (Omura andSato, J. Biol. Chem. 239:2370-2378, 1964), ancymidol (Coolbaugh et al.,Plant Physiol. 62:571-576, 1978), and ketoconazole (Rademacher, Annu.Rev. Plant Physiol. Plant Mol. Biol. 51:501-531, 2000).

Recent results suggest that the hydroxylation of 5-epi-aristolochene isan important regulated step in capsidiol biosynthesis. In studies toevaluate the effectiveness of methyl-jasmonate as an inducer ofcapsidiol biosynthesis in tobacco cell cultures, Mandujano-Chávez et al.(Arch. Biochem. Biophys. 381:285-294, 2000), reported that the modestaccumulation of this phytoalexin was accompanied by a strong inductionof EAS. This result implied that steps before or after the sesquiterpenecyclase reaction were limiting. Using an in vivo assay measuring theconversion rate of radiolabeled 5-epi-aristolochene to capsidiol, a verylimited induction of the hydroxylase activity was observed in cellstreated with methyl jasmonate relative to that in fungalelicitor-treated cells. This result pointed to the hydroxylase reactionsas a potentially limiting step in capsidiol biosynthesis.

SUMMARY OF THE INVENTION

In one aspect, the invention features several isolated cytochrome P450polypeptides (such as CYP71D20, CYP71D21, CYP73A27, CYP73A28, andCYP92A5, and P450s having substantial identity to these polypeptides),as well as isolated nucleic acid molecules that encode these P450s.

In related aspects, the invention features a vector (such as anexpression vector) including an isolated nucleic acid molecule of theinvention and a cell (for example, a prokaryotic cell, such asAgrobacterium or E. coli, or a eukaryotic cell, such as a mammalian,insect, yeast, or plant cell) including the isolated nucleic acidmolecule or vector.

In yet another aspect, the invention features a transgenic plant ortransgenic plant component including a nucleic acid molecule of theinvention, wherein the nucleic acid molecule is expressed in thetransgenic plant or the transgenic plant component. Preferably, thetransgenic plant or transgenic plant component is an angiosperm (forexample, a monocot or dicot). In preferred embodiments, the transgenicplant or transgenic plant component is a solanaceous, maize, rice, orcruciferous plant or a component thereof. The invention further includesa seed produced by the transgenic plant or transgenic plant component,or progeny thereof.

In another aspect, the invention features a method of providing anincreased level of resistance against a disease caused by a plantpathogen in a transgenic plant. The method involves: (a) producing atransgenic plant cell including the nucleic acid molecule of theinvention integrated into the genome of the transgenic plant cell andpositioned for expression in the plant cell; and (b) growing atransgenic plant from the plant cell wherein the nucleic acid moleculeis expressed in the transgenic plant and the transgenic plant is therebyprovided with an increased level of resistance against a disease causedby a plant pathogen.

In another aspect, the invention features a method for producing analtered compound, the method including the steps of contacting thecompound with one or more of the isolated polypeptides disclosed hereinunder conditions allowing for the hydroxylation, oxidation,demethylation, or methylation of the compound and recovering the alteredcompound.

In still another aspect, the invention features a hydroxylating agentincluding any of the isolated polypeptides disclosed herein.

In yet another embodiment, the invention features an isolated nucleicacid molecule that specifically hybridizes under highly stringentconditions to the complement of any one of the sequences described inSEQ ID NO:2 (CYP71D20), SEQ ID NO:4 (CYP71D21), SEQ ID NO:6 (CYP73A27),SEQ ID NO:8 (CYP73A28), or SEQ ID NO:12 (CYP92A5), wherein such anucleic acid molecule encodes a cytochrome P450 polypeptide.

In another aspect, the invention features a host cell expressing arecombinant isoprenoid synthase and a recombinant cytochrome P450. Inpreferred embodiments, the host cell further expresses, independently orin combination, a recombinant acetyltransferase, methyltransferase, orfatty acyltransferase. In other preferred embodiments, the hostexpresses an endogenous or recombinant cytochrome reductase. Preferably,the host cell is a yeast cell, a bacterial cell, an insect cell, or aplant cell.

In a related aspect, the invention features a method for producing anisoprenoid compound, the method including the steps of: (a) culturing acell that expresses a recombinant isoprenoid synthase and a recombinantcytochrome P450 under conditions wherein the isoprenoid synthase and thecytochrome P450 are expressed and catalyze the formation of anisoprenoid compound not normally produced by the cell; and (b)recovering the isoprenoid compound. In preferred embodiments, the hostcell further expresses a recombinant acetyltransferase, a recombinantmethyltransferase, or a recombinant fatty acyltransferase. In otherpreferred embodiments, the host cell expresses an endogenous orrecombinant cytochrome reductase. Preferably, the host cell is a yeastcell, a bacterial cell, an insect cell, or a plant cell.

In yet another aspect, the invention features an isoprenoid compoundproduced according to the above-mentioned methods.

By “P450 polypeptide,” “cytochrome P450,” or “P450” is meant apolypeptide that contains a heme-binding domain and shows a COabsorption spectra peak at 450 nm according to standard methods, forexample, those described herein. Such P450s may also include, withoutlimitation, hydroxylase activity, dual hydroxylase activity, demethylaseactivity, or oxidase activity. Such enzymatic activities are determinedusing methods well known in the art.

By “polypeptide” is meant any chain of amino acids, regardless of lengthor post-translational modification (for example, glycosylation orphosphorylation).

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 80 or 85%, preferably 90%, more preferably 95%, andmost preferably 97%, or even 98% identity to a reference amino acidsequence (for example, the amino acid sequence shown in SEQ ID NOS: 1,3, 5, 7 and 11) or nucleic acid sequence (for example, the nucleic acidsequences shown in SEQ ID NOS:2, 4, 6, 8 and 12, respectively). Forpolypeptides, the length of comparison sequences will generally be atleast 16 amino acids, preferably at least 20 amino acids, morepreferably at least 25 amino acids, and most preferably 35 amino acids.For nucleic acids, the length of comparison sequences will generally beat least 50 nucleotides, preferably at least 60 nucleotides, morepreferably at least 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOXprograms). Such software matches identical or similar sequences byassigning degrees of homology to various substitutions, deletions,and/or other modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

By an “isolated polypeptide” is meant a P450 polypeptide (for example, aCYP71D20 (SEQ ID NO:1), CYP71D21 (SEQ ID NO:3), CYP73A27 (SEQ ID NO:5),CYP73A28 (SEQ ID NO:7), or CYP92A5 (SEQ ID NO:11) polypeptide) that hasbeen separated from components that naturally accompany it. Typically,the polypeptide is isolated when it is at least 60%, by weight, freefrom the proteins and naturally-occurring organic molecules with whichit is naturally associated. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight, a P450 polypeptide. An isolated P450 polypeptide may beobtained, for example, by extraction from a natural source (for example,a plant cell); by expression of a recombinant nucleic acid encoding aP450 polypeptide; or by chemically synthesizing the protein. Purity canbe measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “derived from” or “obtained from” is meant isolated from or havingthe sequence of a naturally-occurring sequence (e.g., cDNA, genomic DNA,synthetic, or combination thereof).

By “isolated nucleic acid molecule” is meant a nucleic acid molecule,e.g., a DNA molecule, that is free of the nucleic acid sequence(s)which, in the naturally-occurring genome of the organism from which thenucleic acid molecule of the invention is derived, flank the nucleicacid molecule. The term therefore includes, for example, a recombinantDNA that is incorporated into a vector; into an autonomously replicatingplasmid or virus; or into the genomic DNA of a prokaryote or eukaryote;or that exists as a separate molecule (for example, a cDNA or a genomicor cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. The term “isolated nucleic acidmolecule” also includes a recombinant DNA which is part of a hybrid geneencoding additional polypeptide sequences.

By “specifically hybridizes” is meant that a nucleic acid sequence iscapable of hybridizing to a DNA sequence at least under low stringencyconditions, and preferably under high stringency conditions. Forexample, high stringency conditions may include hybridization atapproximately 42° C. in about 50% formamide, 0.1 mg/mL sheared salmonsperm DNA, 1% SDS, 2×SSC, 10% Dextran sulfate, a first wash atapproximately 65° C. in about 2×SSC, 1% SDS, followed by a second washat approximately 65° C. in about 0.1×SSC. Alternatively high stringencyconditions may include hybridization at approximately 42° C. in about50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE,1×Denhardt's, followed by two washes at room temperature in 2×SSC, 0.1%SDS, and two washes at between 55-60° C. in 0.2×SSC, 0.1% SDS. Reducingthe stringency of the hybridization conditions may involve lowering thewash temperature and/or washing at a higher concentration of salt. Forexample, low stringency conditions may include washing in 2×SSC, 0.1%SDS at 40° C.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) a P450 polypeptide.

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, forexample, a P450 polypeptide, a recombinant protein, or an RNA molecule).

By “reporter gene” is meant a gene whose expression may be assayed; suchgenes include, without limitation, beta-glucuronidase (GUS), luciferase,chloramphenicol transacetylase (CAT), green fluorescent protein (GFP),beta-galactosidase, herbicide resistant genes, and antibiotic resistancegenes.

By “expression control region” is meant any minimal sequence sufficientto direct transcription. Included in the invention are promoter elementsthat are sufficient to render promoter-dependent gene expressioncontrollable for cell-, tissue-, or organ-specific gene expression, orelements that are inducible by external signals or agents (for example,light-, pathogen-, wound-, stress-, or hormone-inducible elements orchemical inducers such as salicylic acid (SA) or 2,2-dichloroisonicotinic acid (INA)); such elements may be located in the 5′ or 3′regions of the native gene or engineered into a transgene construct.

By “operably linked” is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (for example, transcriptional activator proteins)are bound to the regulatory sequence(s).

By “plant cell” is meant any self-propagating cell bounded by asemi-permeable membrane and typically is one containing a plastid. Sucha cell also requires a cell wall if further propagation is desired.Plant cell, as used herein includes, without limitation, algae,cyanobacteria, seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

By “plant component” is meant a part, segment, or organ obtained from anintact plant or plant cell. Exemplary plant components include, withoutlimitation, somatic embryos, leaves, stems, roots, flowers, tendrils,fruits, scions, and rootstocks.

By “transgene” is meant any piece of DNA which is inserted by artificeinto a cell and typically becomes part of the genome, for example, thenuclear or plastidic genome, of the organism which develops from thatcell. Such a transgene may include a gene which is partly or entirelyheterologous (i.e., foreign) to the transgenic organism, or mayrepresent a gene homologous to an endogenous gene of the organism.

By “transgenic” is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic plants and the DNA (transgene) isinserted by artifice into the nuclear or plastidic genome. A transgenicplant according to the invention may contain one or more engineeredtraits.

By “pathogen” is meant an organism whose infection of viable planttissue elicits a disease response in the plant tissue. Such pathogensinclude, without limitation, bacteria, mycoplasmas, fungi, insects,nematodes, viruses, and viroids. Plant diseases caused by thesepathogens are described in Chapters 11-16 of Agrios, Plant Pathology,3rd ed., Academic Press, Inc., New York, 1988.

By “increased level of resistance” is meant a greater level ofresistance to a disease-causing pathogen in a transgenic plant (or cellor seed thereof) of the invention than the level of resistance relativeto a control plant (for example, a non-transgenic plant). In preferredembodiments, the level of resistance in a transgenic plant of theinvention is at least 20% (and preferably 30% or 40%) greater than theresistance of a control plant. In other preferred embodiments, the levelof resistance to a disease-causing pathogen is 50% greater, 60% greater,and more preferably even 75% or 90% greater than a control plant; withup to 100% above the level of resistance as compared to a control plantbeing most preferred. The level of resistance is measured usingconventional methods. For example, the level of resistance to a pathogenmay be determined by comparing physical features and characteristics(for example, plant height and weight, or by comparing disease symptoms,for example, delayed lesion development, reduced lesion size, leafwilting and curling, water-soaked spots, and discoloration of cells) oftransgenic plants.

By “purified antibody” is meant antibody which is at least 60%, byweight, free from proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, for example, an acquired resistancepolypeptide-specific antibody. A purified P450 antibody may be obtained,for example, by affinity chromatography using a recombinantly-producedP450 polypeptide and standard techniques.

By “specifically binds” is meant an antibody which recognizes and bindsa P450 protein but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample, which naturallyincludes a P450 protein such as CYP71D20, CYP71D21, CYP73A27, CYP73A28,or CYP92A5.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a proposed alternative pathway for thebiosynthesis of capsidiol in elicitor-treated Nicotiana tabacum cells.5-epi-aristolochene is synthesized from FPP by the action of asesquiterpene cyclase, 5-epi-aristolochene synthase (EAS), and issubsequently hydroxylated at C1 and C3 to form capsidiol.

FIG. 2 is a graph showing an induction time course for sesquiterpenecyclase enzyme activity and sesquiterpene hydroxylase activity incellulase-treated cell cultures. Sesquiterpene cyclase(5-epi-aristolochene synthase, EAS) enzyme activity was determined inextracts prepared from control (open squares) and elicitor-treated(closed squares) cells collected at the indicated time points.Sesquiterpene hydroxylase activity was determined using an indirectassay for control (open circles) and elicitor-treated (closed circles)cells. Cell cultures were incubated with [³H]-5-epi-aristolochene for 3hours ending at the indicated time points before quantifying theincorporation of radioactivity into extracellular capsidiol, adihydroxylated form of aristolochene (Mandujano-Chávez et al., Arch.Biochem. Biophys. 381:285-294, 2000).

FIGS. 3A-3B are a series of graphs showing the dose dependent inhibitionof 5-epi-aristolochene hydroxylase activity by ancymidol andketoconazole. Cell cultures were incubated in the presence of cellulase(0.5 μg/mL) plus the indicated concentrations of ancymidol (A) orketoconazole (B) for 12 hours prior to measuring the in vivo5-epi-aristolochene hydroxylase activity in the cell suspension cultures(squares), or the EAS enzyme activity in extracts prepared from thecollected cells (triangles). The in vitro activity of a purified EASpreparation (Back and Chappell, J. Biol. Chem. 270:7375-7381, 1995) wasalso measured at the indicated inhibitor concentrations as an additionaltest for non-specific effects of these inhibitors (circles).

FIG. 4A is a schematic diagram of the primary structure of a generalizedcytochrome P450 with conserved domains used for the design of PCRprimers highlighted (SEQ ID NOS:26-29).

FIG. 4B is a list of the degenerate P450-specific primers (SEQ IDNOS:30-36) that were used in various combinations with vector specificprimers in the amplification of cytochrome P450 cDNA fragments.

FIG. 4C is a scanned image of an ethidium bromide-stained agarose gelshowing the PCR products amplified from a directional cDNA libraryprepared with mRNA isolated from elicitor-treated cells using thedegenerate primer GRRXCP(A/G)— for (SEQ ID NO:35) and the T7vector-specific primer (SEQ ID NO:37). The T3 vector-specific primer isalso shown (SEQ ID NO:38).

FIG. 5 is a series of Northern blots showing the induction time coursefor CYP71D, CYP73A, CYP82E, CYP92A, and EAS transcript accumulation inelicitor treated cells. Total RNA was extracted from tobacco suspensioncells incubated with the cellulase elicitor for the indicated durations,size fractionated by agarose gel electrophoresis under denaturingconditions, and transferred to a nylon membrane before probing with therespective full-length cDNAs. The uniformity of sample loading wasverified by ethidium bromide staining of ribosomal RNA (Loadingcontrol).

FIGS. 6A-6B are a series of graphs showing carbon monoxide (CO)difference spectra of the microsomal fraction isolated from yeastexpressing the CYP92A5 (A) and CYP71D20 (B) cDNAs. Expression of therespective plasmid constructs engineered into the yeast (WAT11) cellswas induced by a galactose treatment, followed by isolation ofmicrosomal preparations. The difference adsorption spectra of microsomesincubated in the presence (solid lines) and absence (broken lines) ofcarbon monoxide was determined.

FIGS. 7A-7D are a series of gas chromatograms of the reaction productsformed upon incubation of microsomes isolated from WAT11 yeast cellscontaining the CYP71D20 expression construct (A and C) or vector controlDNA (B and D) with sesquiterpene substrates. Microsomes isolated fromthe indicated yeast lines were incubated with 5-epi-aristolochene (A andB) or 1-deoxycapsidiol (C and D) in the presence (solid lines) orabsence (dashed lines) of NADPH. The identities of 5-epi-aristolochene,1-dcoxycapsidiol, and capsidiol were verified by mass spectrometry.

FIGS. 8A-8D provide a sequence comparison of the amino acid sequence ofAicotiana tabacum 5-epi-aristolochene (sesquiterpene) hydroxylaseNtCYP71D20 (SEQ ID NO:1) with other plant terpene hydroxylases (SEQ IDNOS:39-43). NrCYP71A5v1 (GenBank accession number CAA70575) catalyzesthe mono-hydroxylation of nerol and geraniol, linear monoterpenes, whilePaCYP71A1 (A35867) catalyzes the epoxidation of these substrates(Hallahan et al., Biochim. Biophys. Acta. 1201:94-100, 1994). MsCYP71D18(AAD44150) and MpCYP71D13 (AAD44151) catalyze the mono-hydroxylation atC6 and C3 of limonene, a cyclic monoterpene, respectively (Lupien etal., Arch. Biochem. Biophys. 368:181-192, 1999). AtCYP701A3 (AAC39505)encodes for kaurene oxidase, which catalyzes a 3-step reaction includinga hydroxylation followed by oxidation of a diterpene (Helliwell et al.,Plant Physiol. 119:507-510, 1999). Shown are sequences from Menthapiperita (MpCYP71D13; SEQ ID NO:39), Mentha spicata (MsCYP71D18; SEQ IDNO:40), Nepeta racemosa (NrCYP71A5v1; SEQ ID NO:41), Nicotiana tabacum(NtCYP71D20; SEQ ID NO:1), Peryea americana (PaCYP71A1; SEQ ID NO:42),and Arabidopsis thaliana (CYP701A3; SEQ ID NO:43). Conserved residuesare shaded.

DETAILED DESCRIPTION

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced byseveral Solanaceous species in response to a variety of environmentalstimuli. It is the primary antimicrobial compound produced by Nicotianatabacum in response to fungal elicitation, and it is formed via theisoprenoid pathway from 5-epi-aristolochene. Much of the biosyntheticpathway for the formation of this compound has been elucidated, exceptfor the enzyme(s) responsible for the conversion of the allylicsesquiterpene 5-epi-aristolochene to its dihydroxylated form, capsidiol.

Accordingly, an in vivo assay for 5-epi-aristolochenehydroxylase-activity was developed and used to demonstrate a dosedependent inhibition of activity by ancymidol and ketoconazole, twowell-characterized inhibitors of cytochrome P450 enzymes. Usingdegenerate oligonucleotide primers designed to the well-conserveddomains found within most P450 enzymes, including the heme bindingdomain, cDNA fragments representing four distinct P450 families (CYP71,CYP73, CYP82, and CYP92) were amplified from a cDNA library preparedagainst mRNA from elicitor-treated cells using PCR. The PCR fragmentswere subsequently used to isolate full-length cDNAs (CYP71D20 (SEQ IDNO:2) and D21 (SEQ ID NO:4), CYP73A27 (SEQ ID NO:6) and A28 (SEQ IDNO:8), CYP82E1 (SEQ ID NO:10), and CYP92A5 (SEQ ID NO:12)), and these inturn were used to demonstrate that the corresponding mRNAs were allinduced in elicitor-treated cells, albeit with different inductionpatterns.

EXAMPLES

There now follows a description of the cloning of several P450s fromNicotiana tabacum. These examples are provided for the purpose ofillustrating the invention, and are not to be considered as limiting.

Inhibition of the 5-epi-aristolochene to Capsidiol Conversion by P450Antagonists

Using an indirect assay, a detailed induction time course of 5EAHactivity in elicitor-induced cell cultures was determined relative tothat of EAS activity (FIG. 2), the well-characterized sesquiterpenecyclase activity that catalyzes the formation of 5-epi-aristolochenefrom FPP (FIG. 1). Using assays for EAS and 5EAH, EAS activity is notdetectable in control cell cultures, but is induced significantly within3 hours and reaches its maximal level within 15 to 18 hours ofelicitor-treatment. Similar to the EAS enzyme activity, 5EAH activitywas negligible in control cell cultures. Nonetheless, after an apparentlag phase of 8 hours, a rapid induction of hydroxylase activity wasobserved 10 to 15 hours post elicitor addition to the cell cultures,reaching a maximum by 18 hours followed by a rather gradual decline of10 to 20% over the next 8 hours.

Tobacco cell suspension cultures treated with cellulase plus varyingconcentrations of ancymidol or ketoconazole were pre-incubated for 12hours before measuring the cells' ability to convert exogenous supplied[³H] labeled 5-epi-aristolochene to radiolabeled capsidiol during asubsequent 3 hour incubation period (FIGS. 3A-3B). Apparent activity of5EAH was inhibited in a dose-dependent manner with approximately 50%inhibition by either 25 μM ancymidol or ketoconazole, and more than 80%by 75 μM ancymidol and 95% by 100 μM ketoconazole (FIGS. 3A and 3B).Importantly, neither the in vitro activity of recombinant EAS nor theinduction of EAS in the elicitor-treated cell cultures was significantlyaffected by ancymidol at concentrations as high as 100 μM (FIG. 3A).Ketoconazole also does not appear to affect the in vitro activity ofEAS. However, the inducibility of cyclase activity in elicitor-treatedcell extracts was inhibited by ketoconazole at concentrations above 50μM (FIG. 3B). Therefore, the specificity of ketoconazole as an inhibitorof P450 type reactions should be assessed at or below a concentration of50 μM under these experimental conditions.

Isolation of Elicitor-Inducible Cytochrome P450 cDNAs

A two-step approach for the isolation of candidate P450 cDNAs wasfollowed. A PCR strategy was first employed using a directional cDNAlibrary prepared against mRNA isolated from elicitor-induced cells asthe template and degenerate PCR primers (FIGS. 4A-4C). Sequencealignments of cytochrome P450s from multiple families across kingdomswere used to identify conserved regions to which a series of degenerateprimers were prepared (FIGS. 4A and 4B). In cloning experiments, 450 to550 bp products were expected from reactions utilizing the primerprepared to the heme-binding domain (GRRXCP(A/G)) (SEQ ID NOS:27 and 28)and the T7 vector primer (FIG. 4C). The mixtures of reaction productswere shotgun cloned, and approximately 100 of the cloned PCR fragmentswere sequenced. About half of the sequenced DNAs contained signaturesequences typical of P450 enzymes as revealed by BlastX databasesearches, and these corresponded to typical plant P450 family members ofthe CYP71, CYP73, CYP92 and CYP82 classes. Each of these PCR fragmentswas isolated multiple times in separate experiments. In addition, weisolated full-length cDNAs for these P450 family members. Table 1compares the similarity and identity of the full-length cDNAs of P450family members with those of their nearest family member in the GenBankdatabase. In addition, FIGS. 8A-8D shows an amino acid alignment ofseveral terpene cytochrome P450s. Alignments were performed using thealgorithm of the MACVECTOR software suite.

TABLE 1 Full-length cDNAs cloned from an elicited cDNA libraryCytochrome P450 Nearest relative/ cDNA clone accession number % Identity% Similarity CYP71D20 CYP71D7 (S. chacoense) 76.5 88.8 Gen EMBL U48435CYP71D21 CYP71D7 (S chacoense) 76.3 88.8 Gen EMBL U48435 CYP73A27CYP73A15 (P. vulgaris) 79.4 92.6 Gen EMBL Y09447 CYP73A28 CYP73A15 (P.vulgaris) 79.2 92.4 Gen EMBL Y09447 CYP82E1 CYP82E1 (N. tabacum) 100.0100.0 Gen EMBL AB015762 CYP92A5 CYP92A3 (N. tabacum) 95.5 98.6 Gen EMBLX96784

The cloned fragments were used in a second step to isolate full-lengthclones from the cDNA library. Screening the cDNA library byhybridization with the CYP71 and CYP73 gene fragments yielded fourfull-length cDNAs, two CYP71Ds and two CYP73As. The former clones weredesignated CYP71D20 and CYP71D21, and the latter were designatedCYP73A27 and CYP73A28. The other two cDNA fragments corresponded totobacco cDNAs already found in the GenBank database, CYP82E1 andCYP92A3. These two cDNAs were cloned using specific primers designedwith the help of the available sequence information to amplify thefull-length cDNA.

Induction of Cytochrome P450 mRNAs in Elicitor-Treated Cells

To correlate a biochemical role for P450s in sesquiterpene metabolism,RNA blot analyses were used to determine the steady-state levels of themRNAs coding for all four of the cytochrome P450 clones and EAS incontrol and elicitor-treated cells (FIG. 5). The mRNAs for all four ofthe P450s were rapidly and transiently induced with slightly differenttime courses relative to one another and to the EAS mRNA. CYP73A27 mRNA,for instance, displayed an induction pattern similar to that of EAS withthe maximum mRNA level occurring 9 to 12 hours after elicitation. Whilethe EAS mRNA remained high throughout the duration of the experiment,the CYP73A27 mRNA was negligible in cells 24 hours afterelicitor-treatment. In contrast, the CYP71D mRNA was more rapidlyinduced than the EAS mRNA, reached its maximum 6 to 9 hours afterelicitation, and was declining by 12 hours when the EAS mRNA level wasstill very high.

Functional Identification of CYP71D20 as 5-epi-aristolochene Hydroxylase

To ascribe functional identity to the various P450 cDNAs, full-lengthcDNAs for CYP71D20, CYP82E1 and CYP92A5 were inserted into the yeastexpression vector pYeDP60 (Urban et al., Biochimie 72:463-472, 1990;Pompon et al., Methods Enzymol. 272:51-64, 1996) and the expression ofeach in WAT11, a yeast line containing an integrated Arabidopsisthaliana cytochrome reductase gene (Pompon et al., Methods Enzymol.272:51-64, 1996; Urban et al., J. Biol. Chem. 272:19176-19186, 1997),was determined. Engineering the CYP73A27 cDNA required an extramodification because of an unusually long N-terminus with severalhydrophilic residues that may interfere with proper intracellulartargeting (Nedelkina et al., Plant Mol. Biol. 39:1079-1090, 1999). Thisunusual leader sequence therefore was replaced with the membraneanchoring sequence of CYP73A1, a cinnamate 4-hydroxylase previouslydemonstrated to express well in yeast (Fahrendorf and Dixon, Arch.Biochem. Biophys. 305:509-515, 1993; Pompon et al., Methods Enzymol.272:51-64, 1996). Expression of all these cDNAs was under the control ofthe glucose-repressible, galactose-inducible GAL10-CYC1 promoter(Guarente et al., Proc. Natl. Acad. Sci. U.S.A. 79:7410-7414, 1982), andexpression was compared to yeast transformed with the parent pYeDP60vector (control) alone.

After induction with galactose for approximately 16 hours, control cellsand cells containing the various P450 constructs were collected, andmicrosomes prepared from each were analyzed for general P450 expressionby CO-difference spectroscopy (Omura and Sato, J. Biol. Chem.239:2370-2378, 1964). Microsomes prepared from cells containing theCYP71D20 (FIG. 6A) and CYP92A5 (FIG. 6B) constructs both showedcharacteristic CO difference spectra with peaks at 450 nm, indicatingthat the encoded proteins were assembling properly with their hemecofactor. Using the extinction coefficient of 91 mM⁻¹·cm⁻¹ for hemebinding proteins (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964),it was determined that approximately 107 pmol of CYP71D20 and 268 pmolof CYP92A5 were expressed in the yeast cells per milligram of totalyeast protein.

Both 5-epi-aristolochene and 1-deoxycapsidiol were metabolized to onlyone product with the same retention time as capsidiol. Obvious by itsabsence, no reaction product having a retention time similar todeoxycapsidiol was detectable in the 5-epi-aristolochene incubations(FIGS. 7A-7D). Co-injection of authentic capsidiol with the respectivereaction products resulted in a single GC peak having a 16.2 minuteretention time, identical to capsidiol. Mass spectra patterns for theseparate reaction products were identical to that for the capsidiolstandard (EIMS m/z 236, 221, 203, 185, 175, 163, 157, 133, 121, 107, 93,79, 67, 55, 43, 41).

The in vivo assay data presented in FIGS. 2 and 3A-3B of the currentwork indicate that the conversion of 5-epi-aristolochene is catalyzed byat least one inducible cytochrome P450 mediated reaction.

Furthermore, any of the cytochrome p450 polypeptides described hereinmay include one or more hydroxylase activities which can incorporatehydroxyl groups into at least two distant sites on an isoprenoidcompound. The addition of these hydroxyl groups may occur, for example,sequentially, by adding a hydroxyl group first to one site and then theother, in either order. Moreover, such hydroxylases may be mutated tolimit their ability to hydroxylate a substrate at only one site, or,alternatively, to provide stereochemical specificity to theirhydroxylating activity.

The above-described experiments were performed using the followingmaterials and methods.

Chemicals

Standard laboratory reagents were purchased from Becton DickinsonMicrobiology Systems (Sparks, Md.), FisherBiotech (Fair Lawn, N.J.) andSigma Chemical Company (St. Louis, Mo.).

Biological Materials and Induction Treatments

Nicotiana tabacum cv. KY14 plants and cell suspension cultures wereused. Cell suspension cultures were maintained in modifiedMurashige-Skoog (Vögeli and Chappell, Plant Physiol. 88:1291-1296,1988). Cultures in their rapid phase of growth (3 days old) were usedfor all experiments. At the indicated times, cells were collected andseparated from media by vacuum filtration and stored at −80° C.

Induction treatments were performed by the addition of the fungalelicitors, cellulase (Trichoderma viride, Type RS, Onozuka) orparaciticein (O'Donohue et al., Plant Mol. Biol. 27:577-586, 1995) atthe indicated concentrations. Paraciticein was purified from E. colicells overexpressing a recombinant paraciticein protein containing acarboxy-terminal histidine purification tag.

In Vivo 5-epi-aristolochene Hydroxylase Assay and Inhibition Studies

5-epi-aristolochene hydroxylase-activity was measured as theincorporation of [³H]-5-epi-aristolochene into extracellular capsidiolby intact cells. [³H]-5-epi-aristolochene was produced by incubating anexcess of [1-³H] farnesyl diphosphate (1 μM, 20.5 Ci/mmol) withrecombinant 5-epi-aristolochene synthase (Back et al., Arch. Biochem.Biophys. 315:527-532, 1994; Rising et al., J. Am. Chem. Soc.122:1861-1866, 2000). The hexane extractable radioactivity fromreactions was treated with a small amount of silica to remove anyfarnesol or residual FPP before quantifying the yield of radioactive5-epi-aristolochene by liquid scintillation counting. The hexane solventwas removed under a gentle stream of N₂ gas, and the dried residue wasre-dissolved in acetone. Control and elicitor-treated cells were thenincubated with [³H]-5-epi-aristolochene (approximately 100,000 dpm at2.5 nM) for 3 hour periods at various points during an induction timecourse before collecting the cell and media samples. Detection andquantification of capsidiol in the extracellular culture media wasperformed as reported previously (Chappell et al., Phytochemistry26:2259-2260, 1987), and the amount of radioactivity incorporated intocapsidiol was determined. For these determinations, samples wereseparated by TLC, and the zones corresponding to capsidiol were scrapedfrom the plate for scintillation counting.

Inhibition studies were performed by the addition of the P450 inhibitorsancymidol (Coolbaugh et al., Plant Physiol. 62:571-576, 1978; Hoshino etal., Phytochemistry 38:609-613, 1995) and ketoconazole (Hoshino et al.,Phytochemistry 38:609-613, 1995; Rademacher, Annu. Rev. Plant Physiol.Plant Mol. Biol. 51:501-531, 2000) directly to the cell cultures orenzyme assay mix. Cell cultures were incubated in the presence ofcellulase (0.5 μg/mL) and indicated concentrations of ancymidol orketoconazole for 12 hours prior to the addition of[³H]-5-epi-aristolochene. After a further 3 hour incubation period, thecells and media were collected. The amount of radioactivity incorporatedinto extracellular capsidiol was determined as described above. Toevaluate secondary effects of these inhibitors, the level of induciblesesquiterpene cyclase activity in the collected cells was determinedaccording to Vogeli et al. (Plant Physiol. 93:182-187, 1990), as well asin vitro assays with purified recombinant EAS (Back et al., Arch.Biochem. Biophys. 315:527-532, 1994) incubated with the indicatedconcentrations of ancymidol and ketoconazole.

All experiments were replicated in several independent trials. While theabsolute values presented may have varied between experiments by as muchas 50%, the trends and time courses were consistent throughout.

Construction of an Elicitor-Induced cDNA Library

Cell cultures were incubated with fungal elicitor (0.5 μg cellulase/mL)for 6 hours before collecting the cells by filtration. The cells werekept frozen at −80° C. until total RNA was extracted from them usingTrizol (Life Technologies, Rockville, Md.) according to themanufacturer's instructions. Poly (A)⁺ RNA was purified by two rounds ofoligo (dT) cellulose column chromatography (Life Technologies,Rockville, Md.). cDNA synthesis and library construction weresubsequently carried out using the UNI-ZAP XR library kit (Stratagene,La Jolla, Calif.), according to manufacturer's instructions.

PCR Cloning Strategy

Cytochrome P450 cDNA fragments were amplified from the elicitor-inducedcDNA library using various combinations of degenerate forward andreverse primers with the vector-specific T3 and T7 primers. The templateDNA was prepared from a 500 μL aliquot of the elicitor-induced cDNAlibrary (3×10⁶ pfu/μL) by heat denaturation at 70° C. for 10 minutes,followed by phenol/chloroform extraction, ethanol precipitation andre-suspension in 500 μL of sterile, deionized water. Amplificationreactions were performed in 50 μL volumes containing 50 mM KCl; 10 mMTris-HCl, pH 8.8; 1.5 mM MgCl₂; 200 μM of each dNTP; 2 μL template DNA;20 pmol each of forward and reverse primer; and 1 unit Taq Polymerase(Life Technologies, Rockville, Md.). Reactions were preheated at 94° C.for 2 minutes, followed by thirty-five cycles of denaturing at 94° C.for 1 minute, annealing at 50° C. for 1 minute 30 seconds, andpolymerization at 72° C. for 2 minutes. The reactions were completed bya 10-minute extension at 72° C. Aliquots of the reaction products wereexamined for DNA products by agarose gel fractionation, and ligateddirectly into the pGEM-T Easy vector (Promega, Madison, Wis.). Resultingrecombinant plasmids containing insert DNAs within the expected sizerange were sequenced using T7 and Sp6 primers.

DNA Sequencing

All the DNA sequencing reactions were performed using the BIGDYE™Terminator Cycle sequencing kit (Perkin-Elmer, Wellesley, Mass.) withthe sequences being read on an automated ABI Prism 310 Genetic Analyzer(Applied Biosystems, Foster City, Calif.). Computer assessment of theDNA sequence information was performed using the MACVECTOR (OxfordMolecular, Madison, Wis.) software package.

cDNA Library Screening

The cDNA library was screened with digoxigenin labeled probes. A 258 bpDNA fragment amplified from the pGEM-deg6.4 clone using gene-specificforward (5′-GGCGGAGAATTTGTCCTGGAATGTCATTTGGTTTAG-3′ (SEQ ID NO:13)) andreverse (5′-GTACAATAGTGAGGTTGACAATG-3′ (SEQ ID NO:14)) primers; and a374 bp DNA fragment amplified from the pBKS-CYPB3.843 clone withspecific forward (5′-GGTGGTTGTGAATGCATG-3′ (SEQ ID NO:15)) and reverse(5′-TTATGCAGCAATAGGCTTGAAGACA-3′ (SEQ ID NO:16)) primers, were used toscreen for CYP71Ds. The probes were labeled with digoxigenin-11-dUTPusing the PCR DIG Labeling Mix (Roche Molecular Biochemicals,Indianapolis, Ind.), hybridized to plaque lifts of the cDNA libraryplated at approximately 10,000 PFUs per 150 mm plate, and washybridization detected with the DIG detection system according to themanufacturer's instructions (Roche Molecular Biochemicals, Indianapolis,Ind.). Plaques exhibiting strong hybridization were plaque purified,auto-subcloned to their plasmid forms according to the manufacturer'srecommendations (Stratagene, La Jolla, Calif.), and then subjected toDNA sequencing as described above.

RNA Analysis

RNA gel blot analysis was carried out using 10 μg aliquots of total RNA.RNA samples were heat-denatured at 70° C. for 15 minutes in samplebuffer (1× MOPS, 50% formamide, 16% formaldehyde, 30% glycerol, and 3%ethidium bromide), and size fractionated on a 1.2% agarose gelcontaining 1×MOPS and 18.1% formaldehyde. Uniformity of sample loadingwas determined by visual inspection of the gel for rRNA bands. The RNAswere then transferred to a Zeta Probe nylon membrane (Bio-RadLaboratories, Hercules, Calif.) and hybridized according to themanufacturer's recommendations. Full-length cDNA probes were labeledwith [³²P]-dCTP (PRIME-IT Kit, Stratagene, La Jolla, Calif.) prior tohybridization. After hybridization, the membranes were washed in2×SSC/0.1% SDS once at room temperature followed by sequential washes in0.2×SSC/0.1% SDS at 42° C. and 65° C. Hybridization was detected with aPhosphoimager (Molecular Dynamics, model 445 SI).

Construction of Yeast Expression Vectors

The coding regions of the P450 cDNAs were cloned into the pYcDP60expression vector (Urban et al., J. Biol. Chem. 272:19176-19186, 1990;Pompon et al., Methods Enzymol. 272:51-64, 1996). Appropriate BamHI,EcoRI, and SstI restriction sites (underlined) were introduced via PCRprimers containing these sequences either upstream of the translationstart site (ATG) or downstream of the stop codon (TAA or TGA). Theprimers used to amplify the CYP71D20 cDNA were5′-GGGGGATCCATGCAATTCTTCAGCTTGGTTTCC-3′ (SEQ ID NO:17) and5′-GGGGAATTCTTACTCTCGAGAAGGTTGATAAGG-3′ (SEQ ID NO:18); for the CYP82E1cDNA 5′-CCCGGATCCATGTATCATCTTCTTTCTCCC-3′ (SEQ ID NO:19) and5′-GGGGAATTCTCAATATTGATAAAGCGTAGGAGG-3′ (SEQ ID NO:20); and for theCYP92A3 cDNA 5′-CCCGGATCCATGCAATCCTTCAGCTTGGTTTCC-3′ (SEQ ID NO:21) and5′-GGGGAGCTCTCACTCGCAAGAAGATTGATAAGG-3′ (SEQ ID NO: 22). Two long,overlapping (italicized) primers5′-GCCATTATCGGCGCAATACTAATCTCCAAACTCCGCGGTAAAAAATTCAAGCTCCCACCTGGTCCAACAGCAGTC-3′ (SEQ ID NO:23) and5′-GGGGGATCCATGGACCTCCTCCTCATAGAAAAAACCCTCGTCGCCTTATTCGCCGCCATTATCGGCGCAATACTA-3′ (SEQ ID NO:24) coding for the N-terminalsequence of CYP73A1 (GenEMBL Z17369) up to the hinge region were usedfor the modification of the membrane anchoring segment of CYP73A27 toavoid possible problems with intracellular targeting due to the unusualN-terminus (Nedelkina et al., 1999); the reverse primer used for bothamplifications was 5′-GGGGAGCTCTTATGCAGCAATAGGCTTGAAGAC-3′ (SEQ IDNO:25). CYP71D20 and CYP73A27 were amplified using full-length cDNAtemplates, whereas CYP82E1 and CYP92A5 were amplified directly from thecDNA library template. Amplifications were performed in 50 μL reactionscontaining 1× Pfx amplification buffer; 1 mM MgSO₄; 300 μM of each dNTP;10 ng template DNA; 20 pmol each of forward and reverse primer; and 1.25units PLATINUM® Pfx Polymerase (Life Technologies, Rockville, Md.).Reactions were preheated at 94° C. for 2 minutes, followed bythirty-five cycles of denaturing at 94° C. for 15 seconds, annealing at55° C. for 30 seconds, and elongating at 68° C. for 1.5 minutes. PCRproducts were ligated into the pGEM-T EASY vector (Promega, Madison,Wis.) and subcloned into the pYcDP60 vector. The resulting constructswere validated by a combination of PCR and DNA sequencing.

Yeast Expression Studies

Verified pYeDP60-P450 cDNA constructs were introduced into the yeastWAT11 line, a derivative of the W303-1B strain (MATa; ade 2-1; his 3-11;leu 2-3, -112; ura 3-1; can^(R); cyr⁺), provided by Dr. P. Urban (Centrede Génétique Moléculaire, CNRS, Gif-sur-Yvette, France). The endogenousNADPH-cytochrome P450 reductase (CPR1) locus has been replaced withATR1, a NADPH-cytochrome P450 reductase from Arabidopsis thaliana(Pompon et al., Methods Enzymol. 272:51-64, 1996; Urban et al., J. Biol.Chem. 272: 19176-19186, 1997), in the WAT11 line. Yeast was grownovernight in a 30° C. shaker in YPAD (1 g/l yeast extract; 1 g/Lpeptone; 20 g/L glucose; 200 mg/L adenine) liquid media. Cultures wereharvested at an A₆₀₀ between 0.5 and 1.5. Cells were collected bycentrifugation at 2,500×g for 5 minutes at 4° C., and resuspended inice-cold, sterile dH₂O. Cells were pelleted again as above andresuspended in 1M sorbitol. Forty μL of yeast suspension was mixed with0.5 to 1 μg plasmid DNA (in <5 μL dH₂O) in a pre-chilled 0.5 mL tube,and transferred to a chilled cuvette with a 0.2 cm electrode gap. Onepulse at 1.5 kV, 25 μF, and 200 Ohms was applied by an EppendorfElectroporator (model 2510). A mixture of 500 μL of YPAD/1M sorbitol wasimmediately added to the electroporated cells. Cells were allowed torecover at 30° C. for 1 hour, then spread onto SGI plates (1 g/lbactocasamino acids; 7 g/l yeast nitrogen base; 20 g/l glucose; 20 mg/ltryptophan; and 20 g/l agar). Transformed colonies appeared after 3 to 6days of incubation at 30° C. Recombinant plasmids were confirmed by PCRassays performed directly on randomly selected yeast colonies.

For expression studies, one colony was added to SGI media (1 g/lbactocasamino acids; 7 g/l yeast nitrogen base; 20 g/l glucose; and 20mg/l tryptophan) and grown at 30° C. for approximately 24 hours. Analiquot of this culture was diluted 1:50 into 250 mL of YPGE (10 g/lbactopeptone; 10 g/l yeast extract; 5 g/l glucose; and 3% ethanol byvolume) and the cells were grown until all glucose was consumed. Theabsence of glucose was determined by placing a 200 μL at aliquot ofculture into a 1.5 mL tube, inserting a DIASTIX urinalysis reagent strip(Bayer, Elkhart, Ind.) for 30 seconds, and observing colorimetricchanges indicating glucose levels. Induction was initiated by theaddition of 5 grams of galactose (final concentration of 2%). Thecultures were maintained at 30° C. for an additional 16 hours beforecollecting the cells by centrifugation at 7,000×g for 10 minutes. Thepelleted cells were washed with 100 mL of TES buffer (50 mM Tris-HCl, pH7.5; 1 mM EDTA; 0.6 M sorbitol). The cells were centrifuged as above,resuspended in 100 mL of TES-M (TES supplemented with 10 mM2-mercaptoethanol), and allowed to incubate at room temperature for 10minutes. The yeast cells were centrifuged again at 7,000×g for 10minutes, and the pellet was resuspended in 2.5 mL extraction buffer (1%bovine serum albumin, fraction V; 2 mM 2-mercaptoethanol; 1 mMphenylmethylsulfonyl fluoride, all dissolved in TES). Glass beads (0.5mm in diameter, Biospec Products, Inc., Bartlesville, Okla.) were addeduntil skimming the surface of the cell suspension. Cell walls weredisrupted manually by hand shaking in a cold room for 10 min at 30second intervals separated by 30 second intervals on ice. Cell extractswere transferred to a 50 mL centrifuge tube, the glass beads were washedthree times with 5 mL of extraction buffer, and the washes were pooledwith the original cell extracts. Microsomes were prepared bydifferential centrifugation at 10,000 g for 10 minutes at 4° C. toremove cellular debris, followed by centrifugation at 100,000×g for 70minutes at 4° C., and microsomal pellets were resuspended in 1.5 mLTEG-M buffer (50 mM Tris-HCl, pH 7.5; 1 mM EDTA; 20% glycerol; and 1.5mM 2-mercaptoethanol) and stored frozen at −80° C. until furtherassayed.

CO Difference Spectra

Fe²⁻.CO vs. Fe²⁺ difference spectroscopy (Omura and Sato, J. Biol. Chem.239:2370-2378, 1964) was performed using 0.4 mL of microsomes suspendedin 1.6 mL of 50 mM Tris-HCl, pH 7.5; 1 mM EDTA; and 20% glycerol. Asmall amount of the reducing agent, sodium dithionite, was added, andthe mixture was distributed between two cuvettes. A baseline wasrecorded between 400 and 500 nm on a Perkin Elmer Lambda 18 UV/visiblespectrophotometer. CO was then bubbled into the sample cuvette for 1minute, and the difference spectrum recorded again. The amount offunctional P450 was estimated based on an absorbance coefficient of 91mM⁻¹·cm⁻¹.

5-epi-aristolochene-1,3-hydroxylase Assays

5-epi-aristolochene-1,3-hydroxylase assays were performed in 0.5 mLpolyethylene tubes in 100 μL volumes. 5-epi-aristolochene or1-deoxycapsidiol dissolved in hexane was added to the tube, and theorganic solvent was removed by incubation of the open tube at 30° C.5-epi-aristolochene and 1-deoxycapsidiol were resuspended in 2 μLdimethyl sulfoxide before adding the reaction mixture. Reactions werecarried out in 100 mM Tris-HCl, pH 7.5, to which microsomal protein wasadded to a final concentration of 1 mg/mL. Reactions were initiated bythe addition of 2 mM NADPH. The final concentration of5-epi-aristolochene and 1-deoxycapsidiol in these assays varied from 20to 50 μM. After incubations for variable lengths of time at 30° C., thereactions were extracted with two volumes of ethyl acetate. The organicextracts were concentrated and evaluated by GC and GC-MS along withstandards of 5-epi-aristolochene (Whitehead et al., Phytochemistry28:775-779, 1989; Rising et al., J. Am. Chem. Soc. 122:1861-1866, 2000),1-deoxycapsidiol (Whitehead et al., Phytochemistry 29:479-182, 1990),and capsidiol (Whitehead et al., Phytochemistry 26:1367-1369, 1987;Milat et al., Phytochemistry 30:2171-2173, 1991). GC analysis wasroutinely performed with an HP5890 GC equipped with a Hewlett-PackardHP-5 capillary column (30 m×0.25 mm, 0.25 μm phase thickness) and FID asdescribed previously (Rising et al., J. Am. Chem. Soc. 122:1861-1866,2000). GC-MS analysis was performed at the University of Kentucky MassSpectrometry Facility using a Varian 3400 gas chromatograph and aFinnigan INCOS 50 quadrupole mass selective detector. The GC wasequipped with a J&W DB-5 ms capillary column (15 m×0.25 mm, 0.25 μmphase thickness) and run with He as the carrier gas (10 psi.). Splitlessinjections were done at an injection port temperature of 280° C. Thecolumn temperature was maintained at 40° C. for 1 minute and thenincreased to 280° C. at 10° C. per minute. Following separation by theGC column, samples were introduced directly into the electron impactionization source. Mass spectra were acquired at 70 eV, scanning from40-440 Da in 1 second.

Production of Cytochrome P450s

Using the standard molecular techniques described herein, the isolationof additional cytochrome P450 coding sequences is readily accomplished.For example, using all or a portion of the amino acid sequence of any ofthe disclosed P450s, one may readily design P450-specificoligonucleotide probes, including P450 degenerate oligonucleotide probes(i.e., a mixture of all possible coding sequences for a given amino acidsequence). These oligonucleotides may be based upon the sequence ofeither DNA strand and any appropriate portion of the P450 nucleotidesequence. General methods for designing and preparing such probes areprovided, for example, in Ausubel et al., 2000, Current Protocols inMolecular Biology, Wiley Interscience, New York, and Berger and Kimmel,Guide to Molecular Cloning Techniques, 1987, Academic Press, New York.These oligonucleotides are useful for P450 gene isolation, eitherthrough their use as probes capable of hybridizing to a P450complementary sequence, or as primers for various amplificationtechniques, for example, polymerase chain reaction (PCR) cloningstrategies.

Hybridization techniques and screening procedures are well known tothose skilled in the art and are described, for example, in Ausubel etal. (supra); Berger and Kimmel (supra); Chen et al. (Arch. Biochem.Biophys. 324:255, 1995); and Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York). Ifdesired, a combination of different oligonucleotide probes may be usedfor the screening of a recombinant DNA library. The oligonucleotides maybe detectably-labeled using methods known in the art and used to probefilter replicas from a recombinant DNA library. Recombinant DNAlibraries are prepared according to methods well known in the art, forexample, as described in Ausubel et al. (supra), or they may be obtainedfrom commercial sources.

As discussed above, P450 oligonucleotides may also be used as primers ina polymerase chain reaction (PCR) amplification cloning strategy. PCRmethods are well known in the art and are described, for example, in PCRTechnology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: AGuide to Methods and Applications, Innis et al., eds., Academic Press,Inc., New York, 1990; and Ausubel et al. (supra). Primers are optionallydesigned to allow cloning of the amplified product into a suitablevector, for example, by including appropriate restriction sites at the5′ and 3′ ends of the amplified fragment (as described herein). Ifdesired, a P450 gene may be isolated using the PCR “RACE” technique, orRapid Amplification of cDNA Ends (see, e.g., Innis et al. (supra)). Bythis method, oligonucleotide primers based on a P450 sequence areoriented in the 3′ and 5′ directions and are used to generateoverlapping PCR fragments. These overlapping 3′- and 5′-end RACEproducts are combined to produce an intact full-length cDNA. This methodis described in Innis et al. (supra) and Frohman et al. (Proc. Natl.Acad. Sci. U.S.A. 85:8998, 1988).

Additional methods for identifying sequences encoding P450s are providedin Maughan et al. (Arch. Biochem. Biophys. 341:104-111, 1997) and Clarket al. (Plant Mol. Biol. 33:875-885, 1997).

Useful P450 sequences may be isolated from any appropriate organism.Confirmation of a sequence's relatedness to a P450 polypeptide disclosedherein may be accomplished by a variety of conventional methods, forexample, by comparing the sequence with a known P450 sequence found in adatabase. In addition, the activity of any P450 may be evaluatedaccording to any of the techniques described herein.

P450 Polypeptide Expression

P450 polypeptides may be produced by transformation of a suitable hostcell with all or part of a P450 DNA (for example, anyone of the P450cDNAs described herein) in a suitable expression vehicle or with aplasmid construct engineered for increasing the expression of a P450polypeptide in vivo.

Those skilled in the field of molecular biology will appreciate that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. The P450 protein may be produced in a prokaryotic host, forexample, E. coli TB 1, or in a eukaryotic host, for example,Saccharomyces cerevisiae, insect cells, mammalian cells (for example,COS 1 or NIH 3T3 cells), or any of a number of plant cells including,without limitation, algae, tree species, ornamental species, temperatefruit species, tropical fruit species, vegetable species, legumespecies, monocots, dicots, or in any plant of commercial or agriculturalsignificance. Particular examples of suitable plant hosts include, butare not limited to, Conifers, Petunia, Tomato, Potato, Tobacco, Grape,Arabidopsis, Lettuce, Sunflower, Oilseed rape, Flax, Cotton, Sugarbeet,Celery, Soybean, Alfalfa, Medicago, Lotus, Vigna, Cucumber, Carrot,Eggplant, Cauliflower, Horseradish, Morning Glory, Poplar, Walnut,Apple, Asparagus, Grape, Rice, Maize, Millet, Onion, Barley, Orchardgrass, Oat, Rye, Tobacco and Wheat.

Such cells are available from a wide range of sources including: theAmerican Type Culture Collection (Rockland, Md.); or from any of anumber of seed companies, for example, W. Atlee Burpee Seed Co.(Warminster, Pa.), Park Seed Co. (Greenwood, S.C.), Johnny Seed Co.(Albion, Me.), or Northrup King Seeds (Harstville, S.C.). Descriptionsand sources of useful host cells are also found in Vasil I. K., CellCulture and Somatic Cell Genetics of Plants, Vol 1, II, ITT; LaboratoryProcedures and Their Applications, Academic Press, New York, 1984;Dixon, R. A., Plant Cell Culture—A Practical Approach, IRL Press, OxfordUniversity, 1985; Green et al., Plant Tissue and Cell Culture, AcademicPress, New York, 1987; and Gasser and Fraley, Science 244:1293, 1989.

For prokaryotic expression, DNA encoding a P450 polypeptide is carriedon a vector operably linked to control signals capable of effectingexpression in the prokaryotic host. If desired, the coding sequence maycontain, at its 5′ end, a sequence encoding any of the known signalsequences capable of effecting secretion of the expressed protein intothe periplasmic space of the host cell, thereby facilitating recovery ofthe protein and subsequent purification. Prokaryotes most frequentlyused are various strains of E. coli; however, other microbial strainsmay also be used. Plasmid vectors are used which contain replicationorigins, selectable markers, and control sequences derived from aspecies compatible with the microbial host. Examples of such vectors arefound in Pouwels et al. (supra) or Ausubel et al. (supra). Commonly usedprokaryotic control sequences (also referred to as “regulatoryelements”) are defined herein to include promoters for transcriptioninitiation, optionally with an operator, along with ribosome bindingsite sequences. Promoters commonly used to direct protein expressioninclude the beta-lactamase (penicillinase), the lactose (lac), thetryptophan (Trp) (Goeddel et al., Nucl. Acids Res. 8:4057, 1980), andthe tac promoter systems, as well as the lambda-derived P.sub.L promoterand N-gene ribosome binding site (Simatake et al., Nature 292:128,1981).

One particular bacterial expression system for P450 production is the E.coli pET expression system (Novagen). According to this expressionsystem, DNA encoding a P450 is inserted into a pET vector in anorientation designed to allow expression. Since the P450 gene is underthe control of the T7 regulatory signals, P450 expression is dependenton inducing the expression of T7 RNA polymerase in the host cell. Thisis typically achieved using host strains which express T7 RNA polymerasein response to IPTG induction. Once produced, recombinant P450 is thenisolated according to standard methods known in the art, for example,those described herein.

Another bacterial expression system for P450 production is the pGEXexpression system (Pharmacia). This system employs a GST gene fusionsystem that is designed for high-level expression of a gene or genefragment as a fusion protein with rapid purification and recovery of thefunctional gene product. The P450 of interest is fused to the carboxylterminus of the glutathione S-transferase protein from Schistosomajaponicum and is readily purified from bacterial lysates by affinitychromatography using Glutathione Sepharose 4B. Fusion proteins can berecovered under mild conditions by elution with glutathione. Cleavage ofthe glutathione S-transferase domain from the fusion protein isfacilitated by the presence of recognition sites for site-specificproteases upstream of this domain. For example, proteins expressed inpGEX-2T plasmids may be cleaved with thrombin; those expressed inpGEX-3X may be cleaved with factor Xa.

Other prokaryotic systems useful for expressing eukaryotic P450s aredescribed by Cooper (Mutat. Res. 454:45-52, 2000) and Dong et al. (Arch.Biochem. Biophys. 327:254-259, 1996). In addition, strategies forenhancing the prokaryotic expression of a cytochrome P450 in combinationwith cytochrome reductase are described in Porter et al. (Drug. Metab.Rev. 31:159-174, 1999).

For eukaryotic expression, the method of transformation or transfectionand the choice of vehicle for expression of the P450 will depend on thehost system selected. Transformation and transfection methods ofnumerous organisms, for example, the baker's yeast Saccharomycescerevisiae, are described, e.g., in Ausubel et al. (supra); Weissbachand Weissbach, Methods for Plant Molecular Biology, Academic Press,1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990; Kindle, K., Proc. Natl. Acad. Sci. U.S.A. 87:1228(1990); Potrykus, I., Annu Rev. Plant Physiol. Plant Mol. Biology 42:205(1991); and BioRad (Hercules, Calif.) Technical Bulletin #1687(Biolistic Particle Delivery Systems). Expression vehicles may be chosenfrom those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987); Gasser and Fraley (supra);Clontech Molecular Biology Catalog (Catalog 1992/93 Tools for theMolecular Biologist, Palo Alto, Calif.); and the references cited above.

One preferred cukaryotic expression system is the mouse 3T3 fibroblasthost cell transfected with a pMAMneo expression vector (Clontech).pMAMneo provides: an RSV-LTR enhancer linked to adexamethasone-inducible MMTV-LTR promoter, an SV40 origin of replicationwhich allows replication in mammalian systems, a selectable neomycingene, and SV40 splicing and polyadenylation sites. DNA encoding a P450is inserted into the pMAMneo vector in an orientation designed to allowexpression. The recombinant P450 is then isolated as described below.Other preferable host cells which may be used in conjunction with thepMAMneo expression vehicle include COS cells and CHO cells (ATCCAccession Nos. CRL 1650 and CCL 61, respectively).

Alternatively, if desired, a P450 is produced by a stably-transfectedmammalian cell line. A number of vectors suitable for stabletransfection of mammalian cells are available to the public, e.g., seePouwels et al. (supra); methods for constructing such cell lines arealso publicly available, e.g., in Ausubel et al. (supra). In oneexample, cDNA encoding the P450 is cloned into an expression vectorwhich includes the dihydrofolate reductase (DHFR) gene. Integration ofthe plasmid and, therefore, the P450-encoding gene into the host cellchromosome is selected for by inclusion of 0.01-300 μM methotrexate inthe cell culture medium (as described in Ausubel et al., supra). Thisdominant selection can be accomplished in most cell types. Recombinantprotein expression can be increased by DHFR-mediated amplification ofthe transfected gene. Methods for selecting cell lines bearing geneamplifications are described in Ausubel et al. (supra); such methodsgenerally involve extended culture in medium containing graduallyincreasing levels of methotrexate. DHFR-containing expression vectorscommonly used for this purpose include pCVSEII-DHrF and pAdD26SV(A)(described in Ausubel et al., supra). Any of the host cells describedabove or, preferably, a DHFR-deficient CHO cell line (for example, CHODHFR cells, ATCC Accession Number CRL 9096) are among the host cellspreferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

A cytochrome P450 may also be produced in insect cells, such cellsinclude, without limitation, Spodoptera frugiperda (Sf)-9, Sf-21, orDrosophila melanogaster Schneider (SL-2) cells. For P450 production,insect cells are typically infected with a baculovirus, for example,Autographa californica Multiple Nuclear Polyhcdrosis Virus (AcMNPV)containing an expression cassette for such a protein, e.g., cytochromeP450, at a multiplicity of infection of 1 to 10. The infected cells aregenerally cultured in a standard insect cell culture medium for 24 to 48hours prior to recovering the protein using standard molecular biologytechniques. If desired, a P450 polypeptide may also be produced ininsect cells directly transfected with a DNA construct containing anexpression cassette encoding the P450.

Furthermore, any of the cytochrome P450s described herein may beproduced in yeast, for example, Pichia pastoris. In order to produce theP450, yeast cells are transformed with an expression cassettecontaining, for example, a promoter such as the AOX1 or phosphoglyceratekinase gene promoter, the P450 gene to be expressed, and a terminator.Such an expression cassette may contain an origin of replication or itmay be integrated into the yeast genomic DNA. The expression cassette isgenerally introduced by lithium acetate transformation or by the use ofspheroplasts. In order to select for successfully transformed cells, theyeast are plated, for example, on minimal media which only allows yeastcarrying the introduced expression cassette to grow.

In addition, expression of recombinant proteins in yeast using aHansenula polymorphs expression system is described in U.S. Pat. Nos.5,741,674 and 5,672,487.

A P450 may also be produced by a stably-transfected plant cell line orby a transgenic plant. Such genetically-engineered plants are useful fora variety of industrial and agricultural applications as discussedbelow. Importantly, this invention is applicable to gymnosperms andangiosperms, and will be readily applicable to any new or improvedtransformation or regeneration method.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants are available to the public;such vectors are described in Pouwels et al. (supra), Weissbach andWeissbach (supra), and Gelvin et al. (supra). Methods for constructingsuch cell lines are described in, e.g., Weissbach and Weissbach (supra),and Gelvin et al. (supra). Typically, plant expression vectors include(1) a cloned P450 gene under the transcriptional control of 5′ and 3′regulatory sequences and (2) a dominant selectable marker. Such plantexpression vectors may also contain, if desired, a promoter regulatoryregion (for example, one conferring inducible or constitutiveexpression, or environmentally- or developmentally-regulated, orpathogen- or wound-inducible, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

The P450 DNA sequence of the invention may, if desired, be combined withother DNA sequences in a variety of ways. The P450 DNA sequence of theinvention may be employed with all or part of the gene sequencesnormally associated with a P450. In its component parts, a DNA sequenceencoding a P450 is combined in a DNA construct having a transcriptioninitiation control region capable of promoting transcription andtranslation in a host cell.

In general, the constructs will involve regulatory regions functional inplants which provide for production of a P450 as discussed herein. Theopen reading frame coding for the P450, or a functional fragmentthereof, will be joined at its 5′ end to a transcription initiationregulatory region such as the sequence naturally found in the 5′upstream region of a P450 structural gene, for example, a CYP71D20 (SEQID NO:2) or CYP71D21 (SEQ ID NO:4) gene. Numerous other transcriptioninitiation regions are available which provide for constitutive orinducible regulation.

For applications when developmental, cell, tissue, hormonal,environmental, or pathogen-inducible expression are desired, appropriate5′ upstream non-coding regions are obtained from other genes; forexample, from genes regulated during seed development, embryodevelopment, leaf development, or in response to a pathogen.

Regulatory transcript termination regions may also be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding a P450 or any convenienttranscription termination region derived from a different gene source.The transcript termination region will contain preferably at least 1-3kb of sequence 3′ to the structural gene from which the terminationregion is derived.

An example of a useful plant promoter according to the invention is acaulimovirus promoter, such as, a cauliflower mosaic virus (CaMV)promoter. These promoters confer high levels of expression in most planttissues, and the activity of these promoters is not dependent on virallyencoded proteins. CaMV is a source for both the 35S and 19S promoters.In most tissues of transgenic plants, the CaMV 35S promoter is a strongpromoter (see, e.g., Odell et al., Nature 313:810, 1985). The CaMVpromoter is also highly active in monocots (see, e.g., Dekeyser et al.,Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389,1990). Moreover, activity of this promoter can be further increased(i.e., between 2-10 fold) by duplication of the CaMV 35S promoter (seee.g., Kay et al., Science 236:1299, 1987; Ow et al., Proc. Natl. Acad.Sci. U.S.A. 84:4870, 1987; and Fang et al., Plant Cell 1:141, 1989).Other useful plant promoters include, without limitation, the nopalinesynthase promoter (An et al., Plant Physiol. 88:547, 1988) and theoctopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989).

For certain applications, it may be desirable to produce the P450 geneproduct in an appropriate tissue, at an appropriate level, or at anappropriate developmental time. For this purpose, there is an assortmentof gene promoters, each with its own distinct characteristics embodiedin its regulatory sequences, which have been shown to be regulated inresponse to the environment, hormones, and/or developmental cues. Theseinclude gene promoters that are responsible for heat-regulated geneexpression (see, e.g., Callis et al., Plant Physiol. 88:965, 1988;Takahashi and Komeda, Mol. Gen. Genet. 219:365, 1989; and Takahashi etal., Plant J. 2:751, 1992); light-regulated gene expression (e.g., thepea rbcS-3A described by Kuhlemeier et al. (Plant Cell 1:471, 1989); themaize rbcS promoter described by Schaffner and Sheen (Plant Cell 3:997,1991); or the chlorophyll a/b-binding protein gene found in peadescribed by Simpson et al. (EMBO J. 4:2723, 1985)); hormone-regulatedgene expression (for example, the abscisic acid (ABA) responsivesequences from the Em gene of wheat described by Marcotte et al. (PlantCell 1:969, 1989); the ABA-inducible HVA1 and HVA22, and the rd29Apromoters described for barley and Arabidopsis by Straub et al. (PlantCell 6:617, 1994), Shen et al. (Plant Cell 7:295, 1994)); andwound-induced gene expression (for example, of wunI described bySiebertz et al. (Plant Cell 1:961, 1989); or organ-specific geneexpression (for example, of the tuber-specific storage protein genedescribed by Roshal et al. (EMBO J. 6: 1155, 1987); the 23-kDa zein genefrom maize described by Schernthaner et al. (EMBO J. 7:1249, 1988); orthe French bean beta-phaseolin gene described by Bustos et al. (PlantCell 1:839, 1989); and pathogen-inducible gene expression described byChappell et al. in U.S. Ser. Nos. 08/471,983; 08/443,639; and08/577,483; hereby incorporated by reference.

Plant expression vectors may also optionally include RNA processingsignals, for example, introns, which have been shown to be important forefficient RNA synthesis and accumulation (Callis et al., Genes and Dev.1:1183, 1987). The location of the RNA splice sequences can dramaticallyinfluence the level of transgene expression in plants. In view of thisfact, an intron may be positioned upstream or downstream of aP450-encoding sequence in the transgene to modulate levels of geneexpression.

In addition to the aforementioned 5′ regulatory control sequences, theexpression vectors may also include regulatory control regions which aregenerally present in the 3′ regions of plant genes (Thornburg et al.,Proc. Natl. Acad. Sci. U.S.A. 84:744, 1987; An et al., Plant Cell 1:115,1989). For example, the 3′ terminator region may be included in theexpression vector to increase stability of the mRNA. One such terminatorregion may be derived from the P1-11 terminator region of potato. Inaddition, other commonly used terminators are derived from the octopineor nopaline synthase signals.

The plant expression vector also typically contains a dominantselectable marker gene used to identify those cells that have becometransformed. Useful selectable genes for plant systems include genesencoding antibiotic resistance genes, for example, those encodingresistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, orspectinomycin. Genes required for photosynthesis may also be used asselectable markers in photosynthetic-deficient strains. Alternatively,the green-fluorescent protein from the jellyfish Aequorea victoria maybe used as a selectable marker (Sheen et al., Plant J. 8:777, 1995; Chiuet al., Current Biology 6:325, 1996). Finally, genes encoding herbicideresistance may be used as selectable markers; useful herbicideresistance genes include the bar gene encoding the enzymephosphinothricin acetyltransferase and conferring resistance to thebroad-spectrum herbicide BASTA (Hoechst AG, Frankfurt, Germany).

Efficient use of selectable markers is facilitated by a determination ofthe susceptibility of a plant cell to a particular selectable agent anda determination of the concentration of this agent which effectivelykills most, if not all, of the transformed cells. Some usefulconcentrations of antibiotics for tobacco transformation include, e.g.,75-100 μg/mL (kanamycin), 20-50 μg/mL (hygromycin), or 5-10 μg/mL(bleomycin). A useful strategy for selection of transformants forherbicide resistance is described, e.g., by Vasil et al., supra.

It should be readily apparent to one skilled in the art of molecularbiology, especially in the field of plant molecular biology, that thelevel of gene expression is dependent, not only on the combination ofpromoters, RNA processing signals, and terminator elements, but also onhow these elements are used to increase the levels of selectable markergene expression.

Plant Transformation

Upon construction of the plant expression vector, several standardmethods are available for introduction of the vector into a plant host,thereby generating a transgenic plant. These methods include (1)Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes)(see, e.g., Lichtenstein and Fuller, In: Genetic Engineering, vol. 6,PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C. P.,and Draper, J., In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford, IRIPress, 1985); (2) the particle delivery system (see, e.g., Gordon-Kammet al., Plant Cell 2:603, 1990; or BioRad Technical Bulletin 1687,supra); (3) microinjection protocols (see, e.g., Green et al., supra);(4) polyethylene glycol (PEG) procedures (see, e.g., Draper et al.,Plant Cell Physiol. 23:451, 1982; or e.g., Zhang and Wu, Theor. Appl.Genet. 76:835, 1988); (5) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353, 1984); (6) electroporationprotocols (see, e.g., Gelvin et al., supra; Dekeyser et al., supra;Fromm et al., Nature 319:791, 1986; Sheen, Plant Cell 2:1027, 1990; orJang and Sheen, Plant Cell 6:1665, 1994); and (7) the vortexing method(see, e.g., Kindle, supra). The method of transformation is not criticalto the present invention. Any method which provides for efficienttransformation may be employed. As newer methods are available totransform crops or other host cells, they may be directly applied.

The following is an example outlining one particular technique, anAgrobacterium-mediated plant transformation. By this technique, thegeneral process for manipulating genes to be transferred into the genomeof plant cells is carried out in two phases. First, cloning and DNAmodification steps are carried out in E. coli, and the plasmidcontaining the gene construct of interest is transferred by conjugationor electroporation into Agrobacterium. Second, the resultingAgrobacterium strain is used to transform plant cells. Thus, for thegeneralized plant expression vector, the plasmid contains an origin ofreplication that allows it to replicate in Agrobacterium and a high copynumber origin of replication functional in E. coli. This permits facileproduction and testing of transgenes in E. coli prior to transfer toAgrobacterium for subsequent introduction into plants. Resistance genescan be carried on the vector, one for selection in bacteria, forexample, streptomycin, and another that will function in plants, forexample, a gene encoding kanamycin resistance or herbicide resistance.Also present on the vector are restriction endonuclease sites for theaddition of one or more transgenes and directional T-DNA bordersequences which, when recognized by the transfer functions ofAgrobacterium, delimit the DNA region that will be transferred to theplant.

In another example, plant cells may be transformed by shooting into thecell tungsten microprojectiles on which cloned DNA is precipitated. Inthe Biolistic Apparatus (Bio-Rad) used for the shooting, a gunpowdercharge (22 caliber Power Piston Tool Charge) or an air-driven blastdrives a plastic macroprojectile through a gun barrel. An aliquot of asuspension of tungsten particles on which DNA has been precipitated isplaced on the front of the plastic macroprojectile. The latter is firedat an acrylic stopping plate that has a hole through it that is toosmall for the macroprojectile to pass through. As a result, the plasticmacroprojectile smashes against the stopping plate, and the tungstenmicroprojectiles continue toward their target through the hole in theplate. For the present invention, the target can be any plant cell,tissue, seed, or embryo. The DNA introduced into the cell on themicroprojectiles becomes integrated into either the nucleus or thechloroplast.

In general, transfer and expression of transgenes in plant cells are nowroutine practices to those skilled in the art, and have become majortools to carry out gene expression studies in plants and to produceimproved plant varieties of agricultural or commercial interest.

Transgenic Plant Regeneration

Plants cells transformed with plant expression vectors can beregenerated, for example, from single cells, callus tissue, or leafdiscs according to standard plant tissue culture techniques. It is wellknown in the art that various cells, tissues, and organs from almost anyplant can be successfully cultured to regenerate an entire plant; suchtechniques are described, e.g., in Vasil (supra), Green et al. (supra),Weissbach and Weissbach (supra) and Gelvin et al. (supra).

In one particular example, a cloned P450, under the control of the EAS4promoter and the nopaline synthase terminator and carrying a selectablemarker (for example, kanamycin resistance), is transformed intoAgrobacterium. Transformation of leaf discs (for example, of tobaccoleaf discs), with vector-containing Agrobacterium is carried out asdescribed by Horsch et al. (Science 227:1229, 1985). Putativetransformants are selected after a few weeks (for example, 3 to 5 weeks)on plant tissue culture media containing kanamycin (e.g., 100 μg/mL).Kanamycin-resistant shoots are then placed on plant tissue culture mediawithout hormones for root initiation. Kanamycin-resistant plants arethen selected for greenhouse growth. If desired, seeds fromself-fertilized transgenic plants can then be sowed in soil-less mediumand grown in a greenhouse. Kanamycin-resistant progeny are selected bysowing surface sterilized seeds on hormone-free kanamycin-containingmedia. Analysis for the integration of the transgene is accomplished bystandard techniques (see, for example, Ausubel et al. (supra); Gelvin etal. (supra)).

Transgenic plants expressing the selectable marker are then screened fortransmission of the transgene DNA by standard immunoblot and DNAdetection techniques. Each positive transgenic plant and its transgenicprogeny is unique in comparison to other transgenic plants establishedwith the same transgene. Integration of the transgene DNA into the plantgenomic DNA is in most cases random, and the site of integration canprofoundly affect the levels and the tissue and developmental patternsof transgene expression. Consequently, a number of transgenic lines areusually screened for each transgene to identify and select plants withthe most appropriate expression profiles.

Transgenic lines are generally evaluated for levels of transgeneexpression. Expression at the RNA level is determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis are employed and include PCR amplification assays usingoligonucleotide primers designed to amplify only transgene RNA templatesand solution hybridization assays using transgene-specific probes (see,e.g., Ausubel et al. (supra)). The RNA-positive plants are then analyzedfor protein expression by Western immunoblot analysis using specificantibodies to the P450 (see, e.g., Ausubel et al., supra). In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using transgene-specific nucleotide probes andantibodies, respectively, to localize sites of expression withintransgenic tissue.

Once the recombinant P450 is expressed in any cell or in a transgenicplant (for example, as described above), it may be isolated, e.g., usingaffinity chromatography. In one example, an anti-P450 antibody (e.g.,produced as described in Ausubel et al., supra, or by any standardtechnique) may be attached to a column and used to isolate thepolypeptide. Lysis and fractionation of P450-producing cells prior toaffinity chromatography may be performed by standard methods (see, e.g.,Ausubel et al., supra). Once isolated, the recombinant protein can, ifdesired, be further purified, for example, by high performance liquidchromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistryand Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

These general techniques of polypeptide expression and purification canalso be used to produce and isolate useful P450 fragments or analogs.

Use

The aforementioned cytochrome P450 polypeptides of the invention areuseful in the biosynthesis of hormones, lipids, and secondarymetabolites, and may also help plants tolerate potentially harmfulexogenous chemicals such as herbicides, pesticides, and pollutants. Inaddition, such cytochrome P450 polypeptides are useful in the chemicaldefense of plants against insects, as well as against bacterial, viral,and fungal infection.

Engineering Plant Disease Resistance

Plasmid constructs designed for the expression of a P450 gene productare useful, for example, for activating plant defense pathways thatconfer anti-pathogenic properties to a transgenic plant, for example,the production of phytoalexins. P450 genes that are isolated from a hostplant (e.g., Nicotiana) may be engineered for expression in the sameplant, a closely related species, or a distantly related plant species.For example, a P450 gene may be engineered for constitutive low-levelexpression and then transformed into a Nicotiana host plant.Alternatively, the P450 gene may be engineered for expression in othersolanaceous plants, including, but not limited to, potato and tomato. Toachieve pathogen resistance, it is important to express a P450 proteinat an effective level. Evaluation of the level of pathogen protectionconferred to a plant by ectopic expression of the P450 gene isdetermined according to conventional methods and assays.

INDUSTRIAL APPLICATIONS

The invention also includes engineering host cells to include novelisoprenoid metabolic pathways useful in the production of new isoprenoidcompounds. By introducing genes encoding an isoprenoid synthase (asdisclosed in U.S. Pat. No. 5,824,774 and WO 00/17327) and a cytochromeP450, an acetyltransferase, a methyl transferase, a fattyacyltransferase, or a combination thereof, various isoprenoid reactionproducts may be modified, controlled, or manipulated, resulting inenhancement of production of numerous isoprenoid reaction products, forexample, the production of novel monoterpenes, diterpenes, andsesquiterpenes. Such compounds are useful as phytoalexins, insecticides,perfumes, and pharmaceuticals such as anti-bacterial and fungal agents.

In one working example, an isoprenoid synthase or a chimeric isoprenoidsynthase (as disclosed in U.S. Pat. No. 5,824,774 and WO 00/17327) and aP450 gene are introduced into yeast, for example, using any of theprocedures described herein. If desired, such cells may also express,either independently or in combination, an acetyltransferase (see, forexample, Walker et al., Proc. Natl. Acad. Sci. U.S.A. 18:583-587, 2000),a methylase transferase gene (see, for example, Diener et al., PlantCell 12:853-870, 2000), or a fatty acyltransferase gene, as well as acytochrome reductase. Cells are then cultured under standard conditionsand the production of isoprenoid compounds is assayed according tomethods known in the art. Isoprenoid compounds are further purifiedaccording to methods well known in the art. Cells expressing novelisoprenoid compounds are taken as useful in the invention.

Such methods provide a unique approach for producing novel isoprenoidstarting materials and end products. Either prokaryotic or cukaryoticcells transformed with any of the aforementioned enzymes (orcombinations thereof) may be used. Moreover, isoprenoid compounds may beproduced in any number of ways known in the art including an in vitrocombination of purified enzymes with an appropriate substrate or directfermentation using a host cell which expresses any combination of theaforementioned enzymes and the appropriate substrates sufficient todrive production of isoprenoid compounds.

The invention is also useful for the production of insect attractantsand deterrents, which may either deter insect pests or attract insectpredators. In addition, the invention is also useful for generatingnovel flavorings and perfumes.

Other Embodiments

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

The invention claimed is:
 1. A method for producing in a recombinanthost cell an isoprenoid compound not endogenously produced by anon-recombinant host cell, comprising: a) providing a recombinant hostcell that comprises heterologous nucleic acid encoding an isoprenoidsynthase, and a heterologous nucleic acid encoding one or moreprotein(s) comprising a CYP71, CYP73, CYP82 or a CYP92 family cytochromeP450 polypeptide, wherein the nucleid acid encoding a CYP71, CYP73,CYP82 or a CYP92 family cytochrome P450 polypeptide can be amplifiedwith degenerate primers based on any one of SEQ ID NOs: 26-29; whereinthe isoprenoid synthase catalyzes production of an isoprenoid compound;wherein the isoprenoid synthase is a diterpene synthase; and wherein thecytochrome P450 polypeptide(s) catalyzes dual hydroxylation, oxidation,demethylation or methylation of the isoprenoid compound; and b)culturing the recombinant host cell under conditions suitable forexpressing the isoprenoid synthase and the cytochrome P450polypeptide(s) under conditions for producing the isoprenoid compound;wherein the synthase and the cytochrome P450 polypeptide(s) catalyzeformation of the isoprenoid compound in the host cell.
 2. The method ofclaim 1, wherein at least one cytochrome P450 polypeptide has oxidaseactivity.
 3. The method of claim 1, wherein at least one cytochrome P450polypeptide has dual hydroxylase activity.
 4. The method of claim 1,wherein the host cell is a yeast cell, a bacterial cell, an insect cellor a plant cell.
 5. The method of claim 1, wherein the host cell is ayeast cell.
 6. The method of claim 5, wherein the yeast is Saccharomycescerevisiae.
 7. The method of claim 1, wherein at least one of thecytochrome P450 polypeptides is 5-epi-aristolochene hydroxylase orkaurene oxidase.
 8. The method of claim 1, wherein the isoprenoidcompound is a diterpene.
 9. The method of claim 1, further comprising c)isolating the isoprenoid compound.
 10. A host cell, comprising nucleicacid encoding an isoprenoid synthase and nucleic acid encoding one ormore protein(s) comprising a CYP71, CYP73, CYP82 or a CYP92 familycytochrome P450 polypeptides encoded by nucleic acid that can beamplified with degenerate primers based on any one of SEQ ID NOs: 26-29,wherein: the nucleic acid encoding the synthase and the nucleic acidencoding the P450 polypeptides are heterologous to the host cell; thesynthase catalyzes production of an isoprenoid compound; the isoprenoidsynthase is a diterpene synthase; the P450 polypeptides catalyzehydroxylation, oxidation, demethylation or methylation of the isoprenoidcompound; at least one of the P450 polypeptide(s) catalyzes the dualhydroxylation, oxidation, demethylation or methylation of the isoprenoidcompound whose production is catalyzed by the synthase; and theisoprenoid compound produced by the host cell is not endogenouslyproduced by a non recombinant host cell.
 11. The host cell of claim 10that is a yeast cell, a bacterial cell, an insect cell or a plant cell.12. The host cell of claim 10 that is a yeast cell.
 13. The host cell ofclaim 10, wherein at least one of the cytochrome P450 polypeptide(s) is5-epi-aristolochene hydroxylase or kaurene oxidase.
 14. The method ofclaim 1, wherein at least one of the cytochrome P450 polypeptide(s) isselected from among polypeptides comprising at least 80% identity to anamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9 and SEQ ID NO:11.
 15. The host cell of claim 10,wherein at least one of the cytochrome P450 polypeptide(s) is selectedfrom among polypeptides comprising at least 80% identity to an aminoacid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9 and SEQ ID NO:11.